key: cord-0310555-f80cfjrn authors: Adenaiye, O. O.; Lai, J.; Bueno de Mesquita, P. J.; Hong, F. H.; Youssefi, S.; German, J. R.; Tai, S.- H. S.; Albert, B. J.; Schanz, M.; Weston, S.; Hang, J.; Fung, C. K.; Chung, H. K.; Coleman, K. K.; Sapoval, N.; Treangen, T.; Maljkovic Berry, I.; Mullins, K. E.; Frieman, M.; Ma, T.; Milton, D. K.; Group, University of Maryland StopCOVID Research title: Infectious SARS-CoV-2 in Exhaled Aerosols and Efficacy of Masks During Early Mild Infection date: 2021-08-13 journal: nan DOI: 10.1101/2021.08.13.21261989 sha: 64a242e298924de01846a17d58b6c4e8e6601491 doc_id: 310555 cord_uid: f80cfjrn Background: SARS-CoV-2 epidemiology implicates airborne transmission; mask source-control efficacy for, variant impact on, and infectiousness of aerosols are not well understood. Methods: We recruited COVID-19 cases to give blood, saliva, mid-turbinate and fomite (phone) swabs, and 30-minute breath samples while vocalizing into a Gesundheit-II, with and without masks at up to two visits two days apart. We quantified and sequenced viral RNA, cultured virus, and assayed sera for anti-spike and anti-receptor binding domain antibodies. Results: We enrolled 61 participants with active infection, May 2020 through April 2021. Among 49 seronegative cases (mean days post onset 3.8 {+/-}2.1), we detected SARS-CoV-2 RNA in 45% of fine ([≥]5 m), 31% of coarse (>5 m) aerosols, and 65% of fomite samples overall and in all samples from four alpha variant cases. Masks reduced viral RNA by 48% (95% confidence interval [CI], 3 to 72%) in fine and by 77% (95% CI, 51 to 89%) in coarse aerosols. The alpha variant was associated with a 43-fold (95% CI, 6.6 to 280-fold) increase in fine aerosol viral RNA that remained a significant 18-fold (95% CI, 3.4 to 92-fold) increase adjusting for viral RNA in saliva, in mid-turbinate swabs, and other potential confounders. Two fine aerosol samples, collected days 2-3 post illness onset, while participants wore masks, were culture-positive. Conclusion: SARS-CoV-2 is evolving toward more efficient airborne transmission and loose-fitting masks provide significant but only modest source control. Therefore, until vaccination rates are very high, continued layered controls and tight-fitting masks and respirators will be necessary. The World Health Organization [1] and U.S. Centers for Disease Control and Prevention[2] recently 63 acknowledged the growing scientific consensus that inhalation exposure is an important route of SARS-64 CoV-2 transmission [3, 4] . The totality of evidence from epidemiologic and outbreak investigations, 65 combined with data on the size distribution of exhaled aerosols and corresponding quantitative models, 66 is compelling [4] . However, culture of the virus from exhaled aerosols, and direct measures of the 67 efficacy of face masks as viral aerosol source control when worn by actual patients have been lacking. 68 Previous reports of infectious virus[5,6] and viral RNA concentrations in room air [7, 8] do not provide a 69 clear picture of how much virus infected persons shed into the air. These gaps lead to uncertainty in 70 estimates of exposure, derived from retrospective analysis of outbreaks [9] . New variants also appear 71 more transmissible, but more quantitative data is needed to discern what that means for implementing 72 effective non-pharmaceutical interventions -still a mainstay of infection protection. 73 74 We collected exhaled breath aerosol samples from polymerase chain reaction (PCR)-confirmed COVID-75 19 cases infected with SARS-CoV-2, including alpha and earlier variants, circulating in a university 76 campus community using a Gesundheit-II (G-II) exhaled breath sampler [10, 11] . We measured 77 concentrations of SARS-CoV-2 RNA and recovered infectious virus from respiratory swabs, saliva, and 78 aerosols, analyzed the effectiveness of face masks as source control, and examined the impact of the 79 alpha variant on aerosol shedding. 80 This study was approved by the University of Maryland Institutional Review Board and the Human 83 Research Protection Office of the Department of the Navy. Electronically signed informed consent was 84 obtained and questionnaire data were collected and stored with REDCap[12] . 85 86 We recruited participants with active infection (defined as positive qRT-PCR for SARS-CoV-2 in 87 respiratory swab or saliva samples) from the University of Maryland College Park campus and 88 surrounding community though a) daily symptom reporting and weekly pooled saliva testing from a 89 cohort of 238 volunteers, b) direct recruiting of recently diagnosed cases targeting local clinics and 90 campus isolation facilities, and c) frequent testing of close contacts of cases for two weeks following last 91 contact. Recruited cases and close contacts completed online consents and a questionnaire (see 92 Supplemental Methods) before in-person confirmation of consent and specimen collection at the 93 University of Maryland School of Public Health. For contacts, we collected a blood specimen for 94 serology at the first visit and measured oral temperature, blood oxygen saturation (SpO2), and collected 95 a mid-turbinate swab (MTS) from each nostril and a saliva sample, at approximately two-day intervals. 96 97 Cohort members with a positive saliva sample, recently-diagnosed cases, and contacts with a positive 98 test during follow-up were invited for viral shedding assessment visits on two days separated by one 99 day. Venous blood was collected at the first visit; MTS, saliva, phone/tablet swab, and 30-minute G-II 100 exhaled breath samples were collected at each visit [10, 11] . We asked participants to provide paired 101 breath samples at each visit with the first collected while wearing a face mask and a second without a 102 mask [13] . We tested the mask brought by the participant and a surgical mask that we provided, and 103 randomized which was tested at the first and second visit to avoid a systematic order bias. Cases studied 104 before September 2020 were asked to repeat the alphabet three times within the 30-minute sampling 105 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted August 13, 2021. ; https://doi.org/10.1101/2021.08.13.21261989 doi: medRxiv preprint period, as previously described [13] . Subsequent cases were asked to shout "Go Terps" 30 times and 106 sing " Happy Birthday" loudly three times at 5, 15, and 25 minutes into each 30-minute sampling period. 107 Participants with more severe symptoms sometimes opted to give only one 30-minute breath sample; for 108 these participants, sampling without a face mask was given priority. test for categorical variables. To handle censored observations below the limit of detection, we applied 151 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 13, 2021. Table 2 ). The frequency of detection of viral RNA in aerosols was 187 greatest 2-5 days post onset of symptoms or first positive test ( Figure S7 ) but was not a significant 188 predictor of the quantity of viral RNA in the aerosols ( . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 13, 2021. ; https://doi.org/10.1101/2021.08.13.21261989 doi: medRxiv preprint (Tables 3 and S3), the alpha variant was associated with significantly greater viral RNA shedding than 197 wild-type and other variants not associated with increased transmissibility. Fine-aerosol shedding 198 remained significantly greater for alpha variant infections (18-fold, 95% CI, 3.4 to 92-fold) after 199 adjusting for the increased viral RNA in MTS and saliva, the number of coughs during sampling 200 sessions, and symptoms (Table 3) . We also analyzed the impact of alpha variants on shedding using the 201 larger data set, including samples collected with and without masks. After controlling for the effect of 202 masks and numbers of coughs during sampling, alpha variant infection was associated with a 100-fold 203 (95% CI, 16 to 650-fold) increase in coarse-and a 73-fold (95% CI, 15 to 350-fold) increase in fine-204 aerosol RNA shedding (Table 3) . 205 206 Effect of masks on viral RNA shedding from seronegative cases 207 We observed statistically significant reductions in aerosol shedding after adjusting for number of coughs 208 during sampling sessions: 77% (95% CI, 51% to 89%) reduction for coarse and 48% (95% CI, 3% to 209 72%) for fine aerosols (Figure 1 ). Analysis of the interaction of masks and the alpha variant ( (Table S4) . We did not 214 observe a significant difference between surgical masks and a composite of the various cloth masks 215 studied (Table S5) . 216 217 Seropositive cases 218 Eight participants had antibodies to SARS-CoV-2 spike protein at the time of breath sample collection. 219 Seropositive cases tended to cough more than seronegative cases ( Table 1 ) but none of the exhaled 220 aerosol samples from seropositive cases had detectable viral RNA (Table S6) . Table S2a ). The RNA 225 concentration associated with a 50% probability of a positive culture was 7.8 x 10 5 for MTS and 5.2 x 226 10 6 for saliva ( Figure S11 , Table S7 ). None of the 75 fine-aerosol samples collected while not wearing 227 face masks were culture-positive. Two (3%) of the 66 fine-aerosol samples collected from participants 228 while wearing face masks were culture-positive, including one from a person infected with the alpha 229 variant 2 days post onset and one from a person with a Nextstrain clade 20G virus 3 days post onset. 230 Fomite and coarse-aerosol samples subjected to culture were negative. 231 Discussion 232 Alpha variant infection yielded one to two orders of magnitude more viral RNA in exhaled breath when 233 compared with earlier strains and variants not associated with increased transmissibility. Our 234 observation of increased aerosol shedding, even after controlling for the increased amounts of viral RNA 235 in the nose and mouth, suggests that evolutionary pressure is selecting for SARS-CoV-2 capable of more 236 efficient airborne transmission. 237 238 We recovered infectious virus from two exhaled breath fine-aerosol samples, approximately two-thirds 239 of MTS, and one-third of saliva samples. Logistic regression analysis of the MTS and saliva samples 240 suggests that there is a small but measurable probability that each RNA copy represents an infectious 241 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 13, 2021. concern or interest, and each case was sampled on only one day. By comparison, our sample included 268 more "wild-type" infections and sampling days per person allowing analysis of the impact of variants on 269 shedding. One delta variant was studied in Singapore and none in this study. The shedding rates detected 270 using the G-II in both studies, however, were lower than those reported by Ma the quality of surgical masks that we could purchase. Therefore, we cannot report on the efficacy of 286 specific loose-fitting masks. This work does, however, provide information on the average amount of 287 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 13, 2021. ; https://doi.org/10.1101/2021.08.13.21261989 doi: medRxiv preprint source control provided by community masking. Finally, logistical considerations required that we 288 freeze samples between collection and culture, with potential loss of infectiousness, especially for dilute 289 aerosol specimens. 290 291 Overall, our results demonstrate that people with mild or asymptomatic SARS-CoV-2 infections 292 released infectious aerosols in their exhaled breath. Face masks provided significant source control 293 suggesting that community-wide masking even with loose-fitting masks can reduce viral aerosols in 294 indoor air by half, making a significant contribution to reducing the spread of COVID-19. Our data also 295 suggest that the virus is evolving toward more effective dissemination through aerosols and demonstrate 296 that infectious virus can escape from loose-fitting masks. With the dominance of newer, more 297 contagious variants than those we studied, increased attention to improved ventilation, filtration, air 298 sanitation, and use of high-quality tight-fitting face masks or respirators (e.g., ASTM F3502-21 face-299 coverings or NIOSH approved N95 filtering face-piece and elastomeric respirators) for respiratory 300 protection will be increasingly important for controlling the pandemic. This will be especially true 301 where vaccination rates are low, vaccine is not available, and for people with poor immune responses or 302 waning immunity. Therefore, our data support community mask mandates and tight-fitting masks or 303 respirators for workers in healthcare but also in all workplaces where people are sharing indoor air or 304 have frequent public contact. 305 306 307 308 Acknowledgements CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 13, 2021. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 13, 2021. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 13, 2021. analyses were controlled for random effects of subject and sample nested within subjects and for censoring by the limit of detection using a linear mixed-effects model for censored responses (R Project lmec-package). a N = Number of participants, n = number of samples for without face mask analysis and number of pairs of same day with and without face masks samples for paired analysis of the effect of face masks. b The adjusted estimates accounted for potential covariates resulting in greater than 10% change in the estimates of the main exposure variable -"Alpha variant" c The effect of mask on samples adjusted for Alpha variant and number of coughs counted during sample collection d Days since start of symptoms or first positive test if asymptomatic or presymptomatic to breath sample; 2 subjects had no symptoms . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted August 13, 2021. ; https://doi.org/10.1101/2021.08.13.21261989 doi: medRxiv preprint FIGURES Figure 1 . Viral RNA shedding in paired with and without face mask samples. Viral RNA measured during 69 same-day paired sampling events with and without mask from 46 seronegative cases. Samples with no detected viral RNA were assigned a copy number value of one. Exhaled breath aerosols were obtained in 30-minute sampling durations. "+mask" = sample collected while wearing a face mask. MTS = mid-turbinate swab, Fomite = swab of participant's mobile phone. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted August 13, 2021. ; https://doi.org/10.1101/2021.08.13.21261989 doi: medRxiv preprint Figure 2 . Viral RNA content and culture results of samples from all sampling events for seronegative cases. Samples with no detected viral RNA were assigned a copy number value of one. Exhaled breath aerosols were obtained during 30-minute sampling events and included unpaired with and without face mask samples. Five fine aerosol samples with face mask and three fomite samples were not available for culture. A subset of MTS, saliva, and coarse aerosol samples were subjected to culture. MTS = midturbinate swab, Fomite = swab of participant's mobile phone. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted August 13, 2021. ; https://doi.org/10.1101/2021.08.13.21261989 doi: medRxiv preprint World Health Organization. Coronavirus disease (COVID-19): How is it transmitted? 2021 SARS-CoV-2 Seroconversion in Humans: A Detailed 369 Protocol for a Serological Assay cov-lineages/pangolin. CoV-lineages Nextstrain SARS-CoV-2 resources R: A Language and Environment for Statistical Computing Elegant Graphics for Data Analysis Efficient Hybrid EM for Linear and Nonlinear Mixed Effects 381 Models with Censored Response Fast Implementation for Normal Mixed Effects Models With Censored Response Estimation of risk due to low doses of microorganisms: a comparison of alternative 387 methodologies SARS-CoV-2 variants reveal features critical for 389 replication in primary human cells Viable influenza A virus in airborne particles from 391 human coughs Respiratory virus shedding in exhaled breath and efficacy 393 of face masks Viral Load of SARS-CoV-2 in Respiratory Aerosols Emitted 395 by COVID-19 Patients while Breathing, Talking, and Singing COVID-19 patients in earlier stages exhaled millions of SARS-CoV-2 per 398 hour Temporal dynamics in viral shedding and transmissibility of COVID-401 19 a Participants with a mid-turbinate or saliva samples positive for SARS-CoV-2 viral RNA by qRT-PCR and seronegative for SARS-CoV-2 spike protein antibody at enrollment and who provided at least one 30-minute sample of exhaled breath. b Number of participants with at least one sample ≥ limit of detection (LOD) or ≥ limit of quantification (LOQ) (Supplementary. c Samples positive and ≥LOD had at least one replicate with confirmed amplification after inspection and quality control (LOD ~75 RNA copies with 95% probability of detection) and ≥LOQ if the mean of replicate assays was ≥250 RNA copies. d GM = geometric mean. The GMs were computed, accounting for samples below the LOD, using a linear mixed-effects model for censored responses (R Project LMEC package) using data for all samples of each sample type with nested random effects of samples within study participant. e The largest quantity of RNA copies detected based on the mean of replicates qRT-PCR aliquots.